I am studying unnatural hydrophobic amino acids and their effect on the stability of T4 lysozyme using free energy perturbation (FEP) methods. The effect of mutating hydrophobic amino acids on protein stability has been an area of great interest since these residues are responsible for two important forces which stabilize proteins: packing in the protein core and the hydrophobic effect. The use of unnatural amino acids allows us to create mutations that specifically vary the amount of nonpolar surface area buried. The project consists of three parts: (1) Design and analysis of unnatural amino acids that replace Leu 133 in the hydrophobic core of T4 lysozyme using computer graphics and molecular dynamics. This part of the project has been completed, and several unnatural amino acid mutants of T4 lysozyme have been created and their stabilities relative to the wild type determined by collaborators at UC Berkeley. (2) FEP studies of two mutations of isolated unnatural amino acids in water: CPE -> ETH and NVL -> MSE. The purpose of these calculations is to determine the methodology required to simulate these mutations accurately. (3) Calculation of relative protein stabilities of the mutants CPE versus ETH and MSE versus NVL. FEP techniques should enable us to better understand how hydrophobic residues stabilize proteins since the relative stability of different mutants can be determined in the native and denatured states. Thus, contributions due to interactions in the native state such as packing can be separated from solvation effects in the denatured state. I use the CGL for viewing protein structures and making slides and photographs for posters, presentations, and publications. These slides and photographs were made by myself by taking pictures of images displayed on the screen.